Project Summary ? Translational regulation has emerged as a key process in the evolution of hepatocellular carcinoma (HCC)1,11. Support of rapid proliferation in cancer requires enhanced protein production as well as gene-specific translational changes that facilitate reprogrammed cellular activities. Recent research has revealed that ribosome composition, including rRNA modification stoichiometry, can affect translational function in cells and bias translation of oncogenic transcripts, altering cell state6,17?20. Although 2% of all rRNA bases are modified, only two of them are N6-methyladenosine (m6A) ? 28S m6A4220 and 18S m6A1832. Our lab previously characterized the 28S m6A4220 methyltransferase (ZCCHC4)21, and we have now biochemically characterized METTL5 (M5) as the 18S m6A1832 methyltransferase, stabilized by the cofactor TRMT112. The function of this site is still unknown, but structural analysis suggests roles in translation initiation and re-initiation events29,31, as it is near the mRNA channel and at the binding site of ribosome recycling factors. Furthermore, our M5 knockout HeLa cells display a markedly hypoproliferative phenotype while overexpression of M5 has been associated with hyperproliferation, including in hepatocellular carcinoma (HCC)8,9,22. We have also shown by imaging and biochemical fractionation that M5 is localized in both the nucleolus and cytosol, suggesting that the 18S m6A site may be dynamically methylated in the cytosol, even after ribosome biogenesis, to regulate and/or fine tune translational processes. The goal of this proposal is to define the functional effects of M5 on translation and investigate the mechanism by which it supports cell proliferation and cancer development. We hypothesize that M5 dynamically methylates m6A1832 in response to oncogenic cell stress with functional consequences in ribosome composition, translational function, and ribosome recycling activities that support proliferation and tumorigenesis. To investigate this hypothesis, we will first thoroughly define the impact of M5 on ribosome composition and function as follows: quantifying M5 and m6A1832 under normal and stress conditions by LC- MS/MS and HPLC; characterizing the effect of M5 on translation through nascent protein synthesis assays, ribosome profiling, and translation reporter assays; and monitoring M5-related changes in stoichiometry of ribosomal proteins and translation-related factors. Then, we will investigate the role and mechanism of M5 in HCC proliferation and tumorigenesis by examining the effects of M5 on ribosome binding and translational activities of ribosome recycling factors, and by evaluating the role of M5 in proliferation of HCC cell lines and tumorigenesis in HCC xenograft mouse models with respect to ribosome recycling processes. Successful completion of this proposal will unveil the function of 18S m6A1832 in translation, clarify the link between M5 and HCC prognosis, potentially guide new translation-based therapy development for HCC, and contribute to our understanding of how dynamic regulation of rRNA modifications can affect the proteome and cellular state in support of cancer development.